scholarly journals Characterization of a novel + 70 Da modification in rhGM-CSF expressed in E. coli using chemical assays in combination with mass spectrometry

Amino Acids ◽  
2021 ◽  
Author(s):  
Magdalena Widgren Sandberg ◽  
Jakob Bunkenborg ◽  
Stine Thyssen ◽  
Martin Villadsen ◽  
Thomas Kofoed
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Covalent Modification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
E Coli ◽  
Gm Csf ◽  
Fragmentation Behavior ◽  
Macrophage Colony

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of different fermentation batches of GM-CSF recombinantly expressed in E. coli, a covalent modification was identified on the protein by intact mass spectrometry. The modification gave a mass shift of + 70 Da and peptide mapping analysis demonstrated that it located to the protein N-terminus and lysine side chains. The chemical composition of C4H6O was found to be the best candidate by peptide fragmentation using tandem mass spectrometry. The modification likely contains a carbonyl group, since the mass of the modification increased by 2 Da by reduction with borane pyridine complex and it reacted with 2,4-dinitrophenylhydrazine. On the basis of chemical and tandem mass spectrometry fragmentation behavior, the modification could be attributed to crotonaldehyde, a reactive compound formed during lipid peroxidation. A low recorded oxygen pressure in the reactor during protein expression could be linked to the formation of this compound. This study shows the importance of maintaining full control over all reaction parameters during recombinant protein production.

Data Mining in Protein Identification by Tandem Mass Spectrometry

2011 ◽  
pp. 472-478
Author(s):  
Haipeng Wang
Keyword(s):  
Mass Spectrometry ◽  
Data Mining ◽  
Tandem Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
Fragmentation Pattern ◽  
Post Translational Modification ◽  
Challenges And Opportunities

Protein identification (sequencing) by tandem mass spectrometry is a fundamental technique for proteomics which studies structures and functions of proteins in large scale and acts as a complement to genomics. Analysis and interpretation of vast amounts of spectral data generated in proteomics experiments present unprecedented challenges and opportunities for data mining in areas such as data preprocessing, peptide-spectrum matching, results validation, peptide fragmentation pattern discovery and modeling, and post-translational modification (PTM) analysis. This article introduces the basic concepts and terms of protein identification and briefly reviews the state-of-the-art relevant data mining applications. It also outlines challenges and future potential hot spots in this field.

scholarly journals Top-Down Proteomic Identification of Shiga Toxin 1 and 2 from Pathogenic Escherichia coli Using MALDI-TOF-TOF Tandem Mass Spectrometry

2019 ◽  
Vol 7 (11) ◽  
pp. 488 ◽  
Author(s):  
Clifton K. Fagerquist ◽  
William J. Zaragoza ◽  
Michelle Q. Carter
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Tandem Mass Spectrometry ◽  
Disulfide Bond ◽  
Shiga Toxin ◽  
Tandem Mass ◽  
Top Down ◽  
E Coli ◽  
B Subunit ◽  
Proteomic Identification

Shiga-toxin-producing Escherichia coli (STEC) are a burden on agriculture and a threat to public health. Rapid methods are needed to identify STEC strains and characterize the Shiga toxin (Stx) they produce. We analyzed three STEC strains for Stx expression, using antibiotic induction, matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) mass spectrometry, and top-down proteomic analysis. E. coli O157:H- strain 493/89 is a clinical isolate linked to an outbreak of hemolytic uremic syndrome (HUS) in Germany in the late 1980s. E. coli O145:H28 strains RM12367-C1 and RM14496-C1 were isolated from an agricultural region in California. The stx operon of the two environmental strains were determined by whole genome sequencing (WGS). STEC strain 493/89 expressed Shiga toxin 2a (Stx2a) as identified by tandem mass spectrometry (MS/MS) of its B-subunit that allowed identification of the type and subtype of the toxin. RM12367-C1 also expressed Stx2a as identified by its B-subunit. RM14496-C1 expressed Shiga toxin 1a (Stx1a) as identified from its B-subunit. The B-subunits of Stx1 and Stx2 both have an intramolecular disulfide bond. MS/MS was obtained on both the disulfide-bond-intact and disulfide-bond-reduced B-subunit, with the latter being used for top-down proteomic identification. Top-down proteomic analysis was consistent with WGS.

scholarly journals Comparison of Online Comprehensive HILIC × RP and RP × RP with Trapping Modulation Coupled to Mass Spectrometry for Microalgae Peptidomics

Separations ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 25 ◽  
Author(s):  
Eduardo Sommella ◽  
Emanuela Salviati ◽  
Simona Musella ◽  
Veronica Di Sarno ◽  
Francesco Gasparrini ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Peptide Mapping ◽  
Reversed Phase ◽  
Peak Capacity ◽  
Tandem Mass ◽  
Two Dimensional ◽  
Mass Spectrometry Detection ◽  
Hydrophilic Interaction

In this work, two online comprehensive two-dimensional liquid chromatography platforms, namely Hydrophilic interaction liquid chromatography × Reversed phase (HILIC × RP) and Reversed phase × Reversed Phase (RP × RP) coupled to mass spectrometry, were compared for the analysis of complex peptide samples. In the first dimension, a HILIC Amide and C18 Bioshell peptide (150 × 2.1 mm, 1.7 and 2.0 μm) columns were selected, while, in the second dimension, a short C18 (50 × 3.0 mm, 2.7 μm) Bioshell peptide column was used. Two C18 trapping columns (10 × 3.0 mm, 1.9 μm), characterized by high retention and surface area, were employed as modulation interface in both HILIC × RP and RP × RP methods. The LC × LC platforms were coupled to UV and tandem mass spectrometry detection and tested for the separation and identification of two gastro-intestinal digests of commercial microalgae formulations (Spirulina Platensis and Klamath). Their performances were evaluated in terms of peak capacity, maximum number and properties of identified phycocyanin peptides. Our results showed that the HILIC × RP approach provided the highest peak capacity values (nc HILIC × RP: 932 vs. nc RP × RP: 701) with an analysis time of 60 min, while the RP × RP approach was able to identify a slight higher number of phycocyanin derived peptides (HILIC × RP: 88 vs. RP × RP: 103). These results point out the flexibility and potential of HILIC × RP and RP × RP based on trapping modulation for peptide mapping approaches.

Mining a Tandem Mass Spectrometry Database To Determine the Trends and Global Factors Influencing Peptide Fragmentation

2003 ◽  
Vol 75 (22) ◽  
pp. 6251-6264 ◽  
Author(s):  
Eugene A. Kapp ◽  
Frédéric Schütz ◽  
Gavin E. Reid ◽  
James S. Eddes ◽  
Robert L. Moritz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Peptide Fragmentation ◽  
Tandem Mass ◽  
Factors Influencing
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